Our work focuses on the use of DNA-based vaccine strategies both as preventive and immunotherapeutic approaches. We have generated efficient SIV and HIV DNA expression vectors. This work is based on our previous recognition that RNA elements (called INS) present within the gag/pol and env coding regions of HIV are responsible for nuclear retention and instability of the transcripts in the absence of Rev, and that these elements can be eliminated by changing the nucleotide composition of the transcripts (RNA optimization; also referred to as codon optimization) without affecting the amino acid sequence. The immunogencity of the antigens was further improved by modifying the trafficking of the antigens. The introduced modifications of the proteins led to more efficient secretion of the SIV antigens resulting in increased cellular and humoral immune responses in the vaccinated mice or rhesus macaques. The immunogenicity of such molecules has been tested in mice and macaques. Studies in mice allowed us to test different DNA vectors and revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine. Different delivery methods of the HIV/SIV antigens are being tested. Another important aspect of HIV vaccine development is the selection of the antigens, which has to take into consideration the diversity of the different HIV clades and the identification of the critical epitopes able to induce relevant immune responses. We are working on optimizing antigens using approaches that use either a conserved epitope approach or mosaic molecules. Using DNA-only vaccination, we found that our optimized DNA vaccine vectors are able to induce potent immune responses able to protect from high viremia in the rhesus macaque/SIV challenge model. A limitation in using DNA as a vaccine is its relative inefficient delivery upon intramuscular injection. Recent developments to improve DNA delivery include in vivo electroporation. We reported that electroporation dramatically increased the efficiency of DNA delivery in both SIV experienced and ART-treated naive rhesus macaques, leading to greatly augmented antigen expression. We found that this vaccination method results in enhanced immune responses with a high frequency of circulating SIV-specific T cells, the presence of multifunctional T cells, and induction of both effector memory and central memory CD4 and CD8 SIV-specific T cells. We reported that the inclusion of IL-12 DNA as adjuvant led to improved quality of the responses. In addition to systemic immune responses, the use of this improved DNA vaccination methodology also induced mucosal responses, albeit only to a subgroup of animals. Although DNA electroporation provides a strong humoral immune response including neutralizing Ab development in macaques, we recently showed that a protein boost can induce higher levels of Ab. Importantly, we showed that injection of DNA and protein either unadjuvanted or adjuvanted in the same muscle at the same time increased Ab production and mucosal dissemination. DNA&Protein co-immunization is superior to vaccination with either of the two individual components in eliciting humoral immune responses. Perhaps more importantly, the vaccine regimen also induced potent long-lasting humoral immunity, detectable for 4 years after the last vaccination. Challenge of animals which received such optimized vaccination regimens showed a significant delay in virus acquisition and improvement in virological control of the highly pathogenic SIV challenge. Thus, efficient DNA delivery methods in combination with improved DNA vaccine cocktails and the inclusion of protein in the vaccine regimen resulted in greatly augmented and more balanced immune responses in vaccinated rhesus macaques. We reported a correlation of systemic and mucosal immune responses and virus acquisition as well as a correlation of cytotoxic cellular immune responses with virus control. An ideal HIV vaccine should provide protection against all HIV-1 variants. HIV sequence diversity and the presence of potential immunodominant decoy epitopes are hurdles in the development of an effective AIDS vaccine. To address these problems, we are exploring approaches to maximize immunological strength and breadth focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant decoy epitopes. We developed a prototype vaccine targeting regions within the p24gag (p24gagCE DNA vaccine). In proof-of-concept studies in mice and macaques, we demonstrated that immunization with this DNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by p55gag DNA vaccination. Importantly, we demonstrated that priming with CE DNA and boosting with p55gag DNA is an effective strategy to maximize responses against Gag, providing a novel concept to increase the magnitude and breadth of vaccination. The translation of this novel concept is currently being pursued in an HVTN/DAIDS-supported clinical trial with the aim to test whether our p24gagCE vaccine develops superior breath and magnitude of gag responses compared to the optimized gag immunogen (p55gag), which showed the highest immune response rate in HVTN clinical trials.